U.S. patent application number 12/295968 was filed with the patent office on 2009-10-01 for anti-allergy compositions.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. Invention is credited to Oded Shoseyov.
Application Number | 20090246287 12/295968 |
Document ID | / |
Family ID | 38141162 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090246287 |
Kind Code |
A1 |
Shoseyov; Oded |
October 1, 2009 |
ANTI-ALLERGY COMPOSITIONS
Abstract
The present invention relates to particles of glucans,
particularly oxidized cellulosed for use in medicine. The invention
further discloses pharmaceutical compositions for the treatment
and/or prophylaxis of diseases or disorders associated with or
mediated by allergens.
Inventors: |
Shoseyov; Oded; (Carmei
Yosef, IL) |
Correspondence
Address: |
BROOKS KUSHMAN P.C.
1000 TOWN CENTER, TWENTY-SECOND FLOOR
SOUTHFIELD
MI
48075
US
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERUSALEM
Jerusalem
IL
|
Family ID: |
38141162 |
Appl. No.: |
12/295968 |
Filed: |
April 10, 2007 |
PCT Filed: |
April 10, 2007 |
PCT NO: |
PCT/IL2007/000450 |
371 Date: |
May 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60789129 |
Apr 5, 2006 |
|
|
|
Current U.S.
Class: |
424/499 ; 424/45;
514/57 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 31/716 20130101 |
Class at
Publication: |
424/499 ; 514/57;
424/45 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/715 20060101 A61K031/715; A61K 9/12 20060101
A61K009/12 |
Claims
1.-46. (canceled)
47. A method for the treatment and/or prophylaxis of at least one
disease or disorder associated with or mediated by at least one
allergen, said method comprising administering to a subject in need
thereof a pharmaceutical composition comprising at least one glucan
selected from oxidized cellulose, pullulan, starch, glycogen,
dextran, lichenin, mannan, galactomannan, arabinoxylan, galacton
and any derivative thereof.
48. The method according to claim 47, wherein said at least one
glucan is oxidized cellulose.
49. The method according to claim 48, wherein said oxidized
cellulose is a salt or derivative of at least one oxidized
cellulose.
50. The method according to claim 49, wherein said oxidized
cellulose, a salt or derivative thereof is in the form of solid
particulates.
51. The method according to claim 50, wherein said solid
particulates are in an amorphous form.
52. The method according to claim 50, wherein said solid
particulates are microparticles or nanoparticles.
53. The method according to claim 50, wherein said solid
particulates have average diameter of between 0.01 and 100
microns.
54. The method according to claim 47, wherein said oxidized
cellulose derivative is a drug derivative.
55. The method according to claim 54, wherein said drug is selected
amongst anti-asthma drugs, anti-allergy drugs, anti-histamine
drugs, smooth muscle cell relaxing agents, mast-cell stabilizers,
anti-IgE drugs, analgesics, hormones, steroids, anti-inflammatory
drugs, antibiotics, anti-viral drugs, anti-bacterial drugs,
anti-fungal drugs, selective or non-selective potassium channel
activators (bronchodilatators), muscarinic M3 receptor antagonists,
M2 receptor agonists, opioid receptor agonists, H3-receptor
agonists, phospholipase A2 inhibitors, 5-lipoxygenase inhibitors,
5-lipoxygenase activating protein (FLAP) inhibitors, leukotriens
modifier drugs, leukotriens receptor antagonists, phosphodiesterase
inhibitors, immunomodulating agents, antibodies against adhesion
molecules, antagonists of tachykinins, mucus secretion inhibitors,
inhaled DNAs, mucus liquefying agents, anti-oxidative agents and
oxygen radical scavengers.
56. The method according to claim 47, wherein said allergen is
selected from weed/plant/tree pollens or spores, animal dander,
house dust mite, dust, lint, mite feces, fungal spores, and
cockroaches.
57. The method according to claim 56, wherein said allergen is
weed, plant or tree pollen.
58. The method according to claim 57, wherein said weed, plant or
tree pollen is selected amongst pollen of dandelion, goldenrod,
nettle, sage, clover, ragweed, mugwort, pellitory, nettles, dock,
Bermuda couch grass, sweet vernal grass, red and blue grasses,
Johnson grass pollen, ryegrass, timothy grass, orchard grass, tall
fescue, meadow fescue and red fescue, alder, oak, ash, cypress,
olive, maple, cedar, western red cedar, elm, birch, hickory,
poplar, American sycamore, walnut, tobacco and cotton.
59. The method according to claim 47, wherein said at least one
disease or disorder associated with or mediated by at least one
allergen is a disease or disorder caused by binding of IgE to an
allergen.
60. The method according to claim 47, wherein said at least one
disease or disorder associated with or mediated by at least one
allergen is a Type I allergic reaction.
61. The method according to claim 47, wherein said at least one
disease or disorder associated with or mediated by at least one
allergen is inflammation and allergic and non-allergic disease or
disorder of the respiratory system or the skin.
62. The method according to claim 60, wherein said disease or
disorder is selected from allergic asthma, asthma, extrinsic
bronchial asthma, chronic obstructive pulmonary disease, hay fever
(seasonal rhinitis), allergic rhinitis, allergic conjunctivitis,
hives, eczema, urticaria, angioedema, onchocercal dermatitis,
atopic dermatitis, dermatitis, swelling, hypersensitivity
pneumonitis and bronchopulmonary dysplasia.
63. The method according to claim 60, wherein said disease or
disorder is selected from allergic rhinitis, allergic
conjunctivitis, hives, hay fever, allergic asthma, asthma,
urticaria, angioedema and atopic dermatitis.
64. The method according to claim 61, wherein said disease or
disorder is allergic asthma, asthma or allergic rhinitis.
65. The method according to claim 47, formulated for topical, oral,
aerosol, intranasal, intraocular, parenteral, subcutaneous,
intravenous, intramuscular, interperitoneal, rectal, or vaginal
administration.
66. The method according to claim 65, being selected from a topical
formulation, an intranasal formulation, and an intraocular
formulation.
67. Particles of oxidized cellulose having an averaged diameter of
between about 0.01 and 100 microns.
68. Nanosized or microsized oxidized cellulose particles suitable
for use in medicine.
69. A method according to claim 47, wherein said pharmaceutical
composition comprising oxidized cellulose particles having an
averaged diameter of between about 0.01 and 100 microns.
70. A method for the treatment and/or prophylaxis of at least one
disease or disorder associated with or mediated by at least one
allergen, said method comprising administering to a subject in need
thereof a pharmaceutical composition comprising oxidized cellulose,
a salt or a derivative thereof.
71. A method for delaying the onset or lessening the severity of at
least one allergic reaction, said method comprising administering
to a subject an effective amount of at least one glucan selected
from oxidized cellulose, pullulan, starch, glycogen, dextran,
lichenin, mannan, galactomannan, arabinoxylan, galacton and any
derivative thereof.
72. A method for reducing a subject's sensitivity to at least one
allergen, said method comprising administering to said subject an
effective amount of at least one glucan selected from oxidized
cellulose, pullulan, starch, glycogen, dextran, lichenin, mannan,
galactomannan, arabinoxylan, galacton and any derivative
thereof.
73. A method for reducing a subject's sensitivity to a pollen
allergen in a subject sensitive to such pollen allergen or a second
allergen immunologically cross-reactive with said pollen allergen,
comprising administering to said subject at least one glucan
selected from oxidized cellulose, pullulan, starch, glycogen,
dextran, lichenin, mannan, galactomannan, arabinoxylan, galacton
and any derivative thereof.
74. The method according to claim 24, wherein said at least one
glucan is oxidized cellulose, a salt or derivative thereof.
75. The method according to claim 28, wherein said oxidized
cellulose, a salt or derivative thereof is in the form of solid
particles.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods and compositions for the
treatment and/or prevention of allergies.
BACKGROUND OF THE INVENTION
[0002] The most powerful and frequent airborne allergens are plant
pollens and among these, grass elicits one of the most widely
spread forms of plant allergens. Inhaled pollen and especially
grass pollen, which contributes to allergic disorders in up to 25%
of adults, represents a major cause of type I allergy response in
to susceptible individuals. Amongst the allergic responses are
allergic rhinitis, conjunctivitis, hives and seasonal asthma. The
impact of type I allergy in industrialized countries has increased
tremendously, especially in children, and is now becoming a major
health problem.
[0003] Asthma is the most common chronic disease of the lung which
affects between 5 and 15% of the population in the industrial
world. It is an inflammatory disease characterized by recurrent
attacks of airway obstruction, traditionally treatable by
anti-inflammatory drugs, particularly steroids. Despite new
treatments, the prevalence and morbidity of asthma have been oil
the rise in the last two decades.
[0004] Asthma is characterized by two phases that occur after
allergen exposure: early asthmatic reaction, EAR, involving early
bronchoconstriction that occurs within minutes, and a late
asthmatic reaction, LAR, involving subsequent inflammatory reaction
of the airways, which occurs 4 to 8 hours later and characterized
by thickening of the bronchial walls due to edema and inflammatory
cell infiltration of lymphocytes, eosinophils and others. The
asthmatic events commence with inhalation of a trigger allergen
that is presented to the immune system. A specific IgE-allergen
complex which forms, subsequently binds to a specific mast cell
membrane receptor (Fc.epsilon.RI), thereby causing degranulation
and release of bronchoconstrictors and pro-inflammatory mediators
like histamine. As a result the airway smooth muscle contracts and
the bronchial lumen becomes narrow, leading to an increase in
airway resistance and shortness of breath. This EAR is self-limited
and resolves spontaneously within 1 hour or by the use of
adrenergic medication. However, after about 4 hours, due to the
inflammatory process initiated by the IgE-allergen complex and the
release of proinflammatory mediators, the bronchial walls become
swollen. This late process (LAR) may be reversed or even
preventable by the use of steroids.
[0005] Asthma, allergic rhinitis and atopic dermatitis are almost
invariably accompanied by elevated levels of IgE. Genetic analyses
of families have shown that bronchial hyperresponsiveness (BHR) and
IgE levels are linked. Thus, in clinical practice, specific
IgE-allergen (as demonstrated by skin testing or in vitro assays)
is generally believed to be inextricably connected to the induction
of allergic airway symptoms, and is used as a guide for
environmental modification and immunotherapy. The interaction of
IgE with antigen is known to lead to a variety of immunological
sequelae. Cross-linking of IgE bound to mast cells by Fc.epsilon.RI
triggers the release of preformed vasoactive mediators, synthesis
of prostaglandins and leukotrienes, and the transcription of
cytokines (proinflammatory mediators).
[0006] The grass allergen of group-I that belong to the
.beta.-expansin gene family shares a high degree of amino acid
sequence similarity with allergens belonging to different groups,
i.e., group II/III, regardless of their origin. Expansins comprise
of two closely related families, .alpha.-expansins (not
glycosylated) and .beta.-expansins (glycosylated). Expansins are
secreted cell wall proteins (.about.26 kDa) and are known to be
involved in the loosening of the plant cell wall during plant
growth as well as in the fruit softening process. The presence of
high levels of expansins in the pollen suggests their involvement
in pollen germination and pollen penetration and growth through the
pistil.
[0007] The expansins are composed of two distinct domains: a
C-terminal cellulose binding domain (CBD) and an N-terminal domain
that exhibits some sequence similarity with the family of 45
endo-glucanases. The CBD allows expansins to interact with the
cellulose microfibrils of the plant cell walls and is known to be
the minimal structure required for plant cell-wall expansion and
disruption of cellulose fiber-to-fiber interactions. It is
important to note that expansins also possess a cystein-proteinase
activity that may further explain their abilities to contribute to
the allergenic reaction.
[0008] Interestingly, the allergens belonging to families II/III
are small proteins (.about.10 kDa) that share .about.40% identity
and .about.60% similarity with the amino acid sequence of the
C-terminal (CBD) of the expansins, thus suggesting that the CBD
part is the common antigen in these groups of proteins (allergen
families I/II/III). A direct support for this observation comes
from the presence of the IgE antigen epitopes specifically on the
cellulose binding cleft of the CBD domain as known for the
rye-grass (Lolium perenne), a major group I allergen Lol pI
protein. In fact, two of the four predicted T-cell epitopes readily
exposed on the surface of the CBD match with IgE-binding regions.
Some of these predicted T-cell epitopes are localized in the
flattened regions of the beta-sandwich exposing aromatic amino
acids that are known to be involved in the recognition of the
cellulose by the CBD part of this protein.
[0009] A further support for this model is given by the fact that
the CBD domain also possesses the classical immunoglobulin-like
folding structure known as the common IgE binding site.
Furthermore, the Ig-like three-dimensional fold of the CBD of the
Lol pI allergen strikingly resembles those of Der f2 and Der p2,
the main group 2 of house dust mite allergens.
[0010] Tree pollens are major causes of pollinosis and among them
olive pollen has high clinical relevance in many areas around the
world. A small olive pollen protein, Ole e 10 (10 kDa) is
recognized as a major inducer of type I allergy in humans. The
ability of Ole e 10 to bind soluble polysaccharides has been known.
Ole e 10 binds specifically to 1,3-.beta.-glucans, in addition this
protein shows sequence identity with the non-catalytic C-terminal
domains of several plant 1,3-.beta.-glucanases (27-53% identity,
44-69% similarity). The change in the secondary structure of Ole e
10 in the presence of laminarin is in agreement with the fact that
CBMs appear to have pre-formed carbohydrate recognition sites that
mirror the solution conformations of their target sugars. The
biochemical activity of Ole e 10 is in agreement with the fact that
callose (1,3-.beta.-glucan) is one of the major component of the
pollen tube wall. Thus, Ole e 10 could act as a
carbohydrate-binding protein that interacts with 1,3-.beta.-glucans
during cell wall synthesis/degradation during pollen
germination.
[0011] Attempts to prevent allergy by employing allergen
inactivating agents for inactivating allergens in the environment
by specific polysaccharides have been documented. US Patent
Applications Nos. 2005/0197319 and 2005/0256082 disclose an
allergen inactivating agent containing a polysaccharide having a
cellulose ether or a starch ether backbone, which is said to be
suitable for inactivation of house dust and other allergens in the
environment by forming a non-specific adsorbing matrix.
[0012] US Patent Application NO. 2004/0082907 discloses an
apparatus for dispensing a restricted amount of powdered materials
particularly to the human nasal tract. This device was used by
Josling et al (2003) and Emberlin et al (2006) for the intranasal
delivery of natural cellulose in the treatment of allergy symptoms
from hay fever, dust mites and animal dander. The authors
hypothesized that the natural crystalline cellulose reacts with the
nasal mucus to create a physical barrier to pollen dust.
[0013] Oxidized cellulose has been investigated as immobilizing
fabric matrices for various agents such as drugs, enzymes and
proteins (Raftery 1980). The release of phenylpropanolamine from an
oxidized cellulose derivative of phenylpropanolamine was also
investigated as having potential drug celivery properties (Zhu,
2004).
[0014] Additionally, microparticles of oxidized cellulose are used
for homeostasis.
REFERENCES
[0015] [1] US Patent Application No. 2005/0197319. [0016] [2] US
Patent Application No. 2005/0256082. [0017] [3] US Patent
Application No. 2004/0082907. [0018] [4] Josling P. and Steadman
S., Use of cellulose powder for the treatment of seasonal allergic
rhinitis. Adv Ther. 2003, 20(4): 213-9. [0019] [5] Emberlin J C.
and Lewis R A., A double blind placebo controlled trial of inert
cellulose powder for the relief of symptoms of Hay fever in adults.
Curr. Med. Res. Opin. 2006, 22(2): 275-85. [0020] [6] Raftery A T.,
Absorbable haemostatic materials and intraperitoneal adhesion
formation. Br. J. Surg. 1980, 67: 57-58. [0021] [7] Zhu L, Kumar V,
Banker G S., Examination of Aqueous Oxidized Cellulose Dispersions
as a Potential Drug Carrier. II. In Vitro and In Vivo Evaluation of
Phenylpropaniolamine Release From Microparticles and Pellets. AAPS
PharmaSciTech 2004, 5(4): 1-8. [0022] [8] Kumar V. and Yang T.,
HNO/HPO-NANO mediated oxidation of cellulose--Preparation and
characterization of bioabsorbable oxidized celluloses in high
yields and with different levels of oxidation. Carbohydrate
Polymers. 2002, 48: 403-12. [0023] [9] Shirai T., Sato A., Chida
K., Hayakawa H I., Akiyama J., Iwata M., Taniguchi M., Reshad K.
and Hara Y., Epigallocatechin gallate-induced histamine release in
patients with green tea-induced asthma. Ann. Allergy Asthma
Immunol., 1997, 79: 65-69. [0024] [10] Hamelmann E., Schwarze J.,
Takeda K., Oshiba A., Larsen G L., Irvin C G., and Gelfand E W.,
Noninvasive measurement of airway responsiveness in allergic mice
using barometric plethysmography. Am. J. Respir. Crit. Care Med.,
1997, 156: 766-775.
SUMMARY OF TUE INVENTION
[0025] The employment of unmodified cellulose in medicaments for
the treatment or prophylaxis of allergic reactions is based on the
assumption that the cellulose, or a derivative thereof, can form a
physical barrier between the allergen and the tissue on which the
cellulose has been applied. Despite the seemingly important
advantages of such a prophylactic, the use of non-modified
crystalline cellulose may be problematic for numerous reasons.
[0026] Plant allergens in general possess natural ability of
interaction with specific polysaccharides of the plant cell-wall
and thus can specifically recognize and bind to target glucans.
Although the natural target glucans for some plant allergen is not
clear, it has been established that plant allergens have none or
very poor recognition of crystalline cellulose, making interaction
with crystalline cellulose substantially impossible. In addition,
the observation that cellulose cannot dissolve in bodily tissues
and thus cannot be regarded as biodegradable or biocompatible
material devoids the use thereof in therapeutically useful
applications.
[0027] The oxidized cellulose, on the other hand, having an
amorphous presentation was found by the inventors of the present
invention to have the necessary recognition of and binding
properties to various plants allergens. The inventors have
successfully determined that particles, e.g., micro or nano in
size, of biocompatible polysaccharides such as glucans may serve as
broad-spectrum allergen blockers by blocking the IgE epitopes. It
is known that the particles, being water-insoluble, can be cleared
from the airway by the mucociliary transport. The structural
plasticity of the various glucans, and particularly that of
oxidized cellulose, on one hand, and the biocompatibility of the
glucans, on the other, make this class of compounds an ideal
broad-spectrum allergen blocker that may be formulated for
administration via inhalation, intranasal application (e.g., as
nasal spray), ocular application (e.g., eye drops), topical
application (e.g., creams, ointments) and for mucosal
application.
[0028] Cellulose and oxidized cellulose are different compounds
having vastly different chemical and physical properties, despite
the fact that the latter may be produced from the former, in terms
of their respective chemical structure, reactivity and toxicity. A
person skilled in the art would appreciate that when searching for
an alternative active agent to commonly used agent, particularly
for human and animal use, one needs to tale into account that the
alternative substance used in the pharmaceutical product must be
toxicologically acceptable, well tolerated by the tissue to which
it is applied, (e.g., skin, mucosa, etc.) stable, and inexpensive
to produce. In the absence of a clear and predictable relationship
to between the action required and the structure of the chemical
agent, its toxicological acceptability, tolerance and/or the
stability, the search for such suitable alternative that possess
the required characteristics is complex. As may be appreciated from
the present description, the aim of the present invention was to
attain an active compound, an alternative to cellulose, which would
be at least as active as commercially available anti-allergic
formulations but at the same time be absent of at least some of the
numerous drawbacks associated with the use of cellulose as
described above.
[0029] In its broadest scope, the present invention relates to the
use of anti-allergy compounds, particularly glucans for preparing
medicaments for therapeutic treatment and/or prophylaxis of various
allergen-mediated or associated disease or disorder, to
pharmaceutical compositions comprising them and to methods for
therapeutic treatment and/or prophylaxis of various
allergen-mediated or associated diseases or disorders. The present
invention also relates to kits or commercial packages containing
any one of the compositions of the invention as particular
formulations and dosage forms and instructions for use.
[0030] In one aspect of the present invention there is provided a
pharmaceutical composition comprising at least one glucan for the
treatment and/or prophylaxis of at least one disease or disorder
associated with at least one allergen, wherein said at least one
glucan is not cellulose or any non-oxidized form thereof. Excluded
from the scope of the present invention are any non-oxidized
cellulose derivatives including ethers, esters, and alkyls
thereof.
[0031] The term "glucan" refers to a polysaccharide of sugar
monomers linked together by glycosidic bonds. The glucan may be
.alpha.- or .beta.-glucan and may be of natural, synthetic or
semi-synthetic origin. The glucan may also be a combination of two
or more glucans. Within the scope of the present invention, the
term does not encompass cellulose or any non-oxidized form thereof
(i.e., cellulose ethers, cellulose esters, etc.), unless
specifically disclosed.
[0032] In one embodiment, the glucan employed by any of the methods
or compositions of the invention is a polysaccharide associated
with the cell walls of pollen and plant pistils of plants.
[0033] In another embodiment, the glucan is selected amongst
polysaccharides that bind protein allergens.
[0034] In another embodiment, the glucan is selected amongst
polysaccharides that upon binding to an allergen interfere with IgE
interactions.
[0035] In yet another embodiment, the glucan is selected amongst
polysaccharides having repeating glucose monomers.
[0036] In another embodiment, the glucan is selected amongst
polysaccharides having a plurality of D-glucose monomers linked
together by glycosidic bonds.
[0037] In still another embodiment, the glucan is selected from
.beta.-1,4-glucans, .beta.-1,3-glucans, .beta.-1,6-glucans,
.alpha.-1,4-glucans, .alpha.-1,6-glucans,
.beta.-1,3/.beta.-1-6-glucans, and
.alpha.-1,4/.alpha.-1,6-glucans.
[0038] In yet another embodiment, the glucan is selected from
oxidized cellulose, pullulan, starch, glycogen, dextran, lichenin,
mannan, galactomannan, arabinoxylan, galacton, chitosan, chitin and
any derivative thereof.
[0039] In another embodiment, the glucan is hemicellulose.
[0040] In another embodiment, the glucan is different from
hemicellulose or cellulose.
[0041] The glucans employed in the present invention may be fully
oxidized partially oxidized or non-oxidized. As stated above, the
glucan, however, is not cellulose or any non-oxidized form
thereof.
[0042] Non-limiting examples of glucans in accordance with the
invention are arabinoxylan, barely beta-glucan, oat beta-glucan
galacton, pullulan, carob galactomannan, xyloglucan, guar
galactomannan, pectic galactan, rhamnogalacturonan-galacturonic
acid, pachyman, curdlan, chi tin derivatives, chitosan, oxidized
cellulose and mannan.
[0043] In another embodiment, the glucan is oxidized cellulose, any
salt and derivative thereof as disclosed herein.
[0044] The oxidized cellulose independently of its crystallinity or
conjugation to another moiety, e.g., drug, as discussed herein, is
typically in the form of solid particulates which may be spherical
or randomly shaped and which may be micro or nano in size
(microparticles and/or nanoparticles). Preferably, the particles of
oxidized cellulose have averaged diameters in the range of 0.01 to
100 microns.
[0045] Thus, in another aspect of the present invention, there is
provided microparticles and/or nanoparticles of oxidized
cellulose.
[0046] In one embodiment, the microparticles and/or nanoparticles
of the oxidized cellulose are suitable for use in medicine.
[0047] The invention additionally provides a pharmaceutical
composition comprising microparticles and/or nanoparticles of
oxidized cellulose, a salt or a derivative thereof.
[0048] The terms "microparticles" and "nanoparticles" are used in
plural merely to indicate that the compositions of the invention
comprise a plurality of such particles and should not be considered
to render the singular form uninventive.
[0049] In one embodiment, the oxidized cellulose particles have an
averaged diameter of between about 0.01 and about 100 microns.
[0050] In another embodiment, the oxidized cellulose particles have
an averaged diameter of between about 0.01 and about 95, 90, 85,
80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or 30 microns.
[0051] In another embodiment, the oxidized cellulose particles lave
in averaged diameter of between about 0.1 and about 50 microns.
[0052] In yet another embodiment, the oxidized cellulose particles
have an averaged diameter of between about 0.1 and about 30
microns.
[0053] In still another embodiment, the oxidized cellulose
particles have an averaged diameter of between about 0.1 and about
10 microns.
[0054] It should be noted that the averaged diameter of the
oxidized cellulose particles may be measured by any method known to
a person skilled in the art. The term "averaged diameter." refers
to the arithmetic mean of measured diameters, wherein the diameters
range .+-.25% of the mean. For example, the expression "averaged
diameter of between about 0.01 and about 100 microns" encompasses
particles having diameters 25% smaller than 0.01 microns and 25%
larger than 00 microns, namely from 0.0075 microns to 125 microns.
An averaged diameter of 30 microns thus refers to an actual average
of between 22.5 and 37.5 microns.
[0055] In another embodiment of the present invention, the
composition comprising microparticles and/or nanoparticles of
oxidized cellulose is suitable for the treatment and/or prophylaxis
of at least one disease or disorder.
[0056] In another embodiment, said disease or disorder is
associated with or mediated by at least one allergen.
[0057] In another aspect of the present invention, there is
provided a pharmaceutical composition comprising oxidized
Cellulose, a salt or a derivative thereof for the treatment and/or
prophylaxis of at least one disease or disorder associated with or
mediated by at least one allergen.
[0058] As known to a person skilled in the art, "Oxidized
cellulose" is a rigid, unbranched, long chain polymer, consisting
of 3,000 to 5,000 glucose residues in .beta.-(1,4) linkage, having
at least part or all of the hydroxymethylene (exocyclic
--CH.sub.2OH) groups oxidized to carboxylic acid (--COOH) groups or
charged carboxylate groups (--COO.sup.-). The oxidized cellulose
employed in the present invention may be synthetic, semi-synthetic
or commercially attained. The oxidized cellulose may also be in its
crystalline form, amorphous form or may be partially crystalline
and partially amorphous.
[0059] In one embodiment, the oxidized cellulose is amorphous
oxidized cellulose.
[0060] In another embodiment, the oxidized cellulose is in the form
of microparticles.
[0061] In another embodiment, the oxidized cellulose is in the form
of nanoparticles.
[0062] Oxidation of cellulose may be achieved by various synthetic
pathways as may be known to the artisan (see for example Kumar et
al, 2002). Such oxidation, preferably does not substantially affect
the glucose ring structure, although a certain degree of ring
opening may occur depending on the oxidative conditions employed.
The degree of oxidation of the hydroxymethylene groups may be
quantified (for example by titration) and the percent weight of the
--COOH groups from the total weight of the polymer (or percent
oxidation) may be calculated. On average, the percent weight of
--COOH was 20% of the total weight of the oxidized cellulose.
[0063] In one embodiment, the % percent weight of the --COOH groups
is at least 3% of the total weight of the oxidized cellulose.
[0064] In another embodiment, the % weight of the --COOH groups of
the total weight of the oxidized cellulose is between 3 and
25%.
[0065] The microparticles and/or nanoparticles of oxidized
cellulose may be prepared by any method known suitable for the
reduction of particle size. Preferably, the microparticles and/or
nanoparticles of oxidized cellulose are prepared by milling the
already oxidized cellulose to a desired particle size, typically in
the range of about 0.01 and 100 microns. Smaller and larger
particles have been obtained. The determination of particles size
as well as of shape may be achieved, as disclosed hereinbelow, by
any measuring technique known to an artisan, such as light and
electron microscopies, X-ray diffraction, etc.
[0066] Thus the present invention further provides a method for the
preparation of microparticles and/or nanoparticles of oxidized
cellulose said method comprising obtaining oxidized cellulose and
affecting a reduction in particle size to the desired size.
[0067] In one embodiment, the oxidized cellulose is in the form of
solid particulates.
[0068] In another embodiment, the reduction in particle size
immediately follows the production of the oxidized cellulose.
[0069] In another embodiment, the oxidized cellulose is
commercially obtained.
[0070] In another embodiment, the reduction in particle size is
achieved by milling.
[0071] In yet another embodiment, the reduction in particle size
affords a mixture of microparticles and nanoparticles of oxidized
cellulose.
[0072] In still another embodiment, the reduction in particle size
affords microparticles.
[0073] In still another embodiment, the reduction in particle size
affords nanoparticles.
[0074] In still another embodiment, the manufactured microparticles
or nanoparticles are further chemically transformed to a salt or a
derivative of said oxidized cellulose.
[0075] The oxidized cellulose may be used in the composition of the
invention in one or more of the following forms:
[0076] (a) acidic form, having substantially all carboxylic groups
protonated, namely in the form of --COOH,
[0077] (b) salt form, having some or all of the oxidized groups in
the charged carboxylate form, namely in the form of --COOX, wherein
X is a monovalent, divalent or multivalent metal ion selected for
example amongst alkali and alkaline metal ions,
[0078] (c) derivatized form, having some or all of the oxidized
groups in the form --COOR, wherein R is an organic radical selected
amongst substituted or unsubstituted C1-C20 alkyl, cycloalkyl,
alkylene or cycloalkylene; substituted or unsubstituted C6-C12 aryl
or arylene; substituted or unsubstituted C5-C12 heteroaryl or
heteroarylene (having at least one heteroatom selected from N, O,
S), C2-C20 alkenyl, alkenylene, cycloalkenyl or cycloakenylene;
wherein each of said groups may be substituted by one or more
organic or inorganic atom or (groups such as halogens (Br, Cl, I,
F), nitro, amines (primary, secondary or tertiary), alkyls, aryls
and others as may be known to a person skilled in the art. The
chemical transformation to the derivatized form, from the acid or
salt forms, may be achieved by any transformation known to the
person skilled in the art. In this respect, see for example
"Comprehensive Organic Functional Group Transformations, Second
Ed., by Alan R. Katritzky and Richard J. K. Taylor, 2004.
[0079] The bond between the O atom of the carboxylic moiety of the
oxidized cellulose and the atom of the R group may be an ionic bond
(such as in the case of a salt of metal or non-metal ions such as
ammoniums) a covalent bond, a coordination bond or any other
interaction which is capable of holding the two moieties--the
oxidized cellulose and the R moiety--in close proximity. In one
embodiment, the bond is a covalent bond. In another embodiment, the
covalent bond is a hydrolysable bond.
[0080] In some embodiment, the oxidized cellulose is a derivatized
oxidized cellulose having at least 0.05% of its oxidized groups
bonded (ironically, covalently, via coordination, etc.) to at least
one R moiety being at least one pharmaceutical (drug). Such drugs
may be selected from anti-asthma drugs, anti-allergy drugs,
antihistamine drugs, smooth muscle cell relaxing agents (e.g.,
linalool, magnesium sulfate), mast-cell stabilizers, anti-IgE
drugs, analgesics, hormones, steroids, anti-inflammatory drugs,
antibiotics, anti-viral drugs, anti-bacterial drugs, anti-fungal
drugs, selective or non-selective potassium channel activators
(bronchodilatators), muscarinic M3 receptor antagonists, M2
receptor agonists, opioid receptor agonists, H3-receptor agonists
(inhibit acetylcholine release), phospholipase A2 inhibitors,
5-lipoxygenase inhibitors, 5-lipoxygenase activating protein (FLAP)
inhibitors, leukotriens modifier drugs, leukotriens receptor
antagonists, phosphodiesterase inhibitors, immunomodulating agents
(e.g., ciclosporine), antibodies against adhesion molecules,
antagonists of tachykinins, mucus secretion inhibitors, inhaled
DNAs (Dronase) and other mucus liquefying agents, anti-oxidative
agents and oxygen radical scavengers.
[0081] Non-limiting examples of such drugs are dexamethasone,
triamcinolone acetonide, beclomethasone, dipropionate, flunisolide,
fluticasone propionate, prednisone, methylprednisolone, mometasone
furoate, chlorcyclizine, chlorpheniramine, triprolidine,
diphenhydramine hydrochloride, fexofenadine hydrochloride,
hydroxyzine hydrochloride, loratadine, promethazine hydrochloride,
pyrilamine, omalizumab, albuterol, pirbuterol, epinephrine,
racepinephrine, adrenaline, isoproterenol, salmeterol,
metaproterenol, bitolterol, fenoterol, formoterol, isoetharine,
procaterol, penicillin G, ampicillin, methicillin, oxacillin,
amoxicillin, cefadroxil, ceforanid, cefotaxime, ceftriaxone,
doxycycline, chlortetracycline, minocycline, tetracycline,
amikacin, gentamycin, kanamycin, neomycin, streptomycin,
netilmicin, paromonmycin, tobramycin, azithromycin, clarithromycin,
erythromycin, ciprofloxacin, lomefloxacin, norfloxacin,
chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin,
vancomycin, aztreonam, clavulanic acid, imipenem, polymyxin,
bacitracin, amphotericin, nystatin, nevirapine, delavirdine,
efavirenz, saquinavir, ritonavir, indinavir, nelfinavir,
amprenavir, zidovudine, stavudine, larnivudine, didanosine,
zalcitabine, abacavir, acyclovir, penciclovir, valacyclovir,
ganciclovir, 1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,
9,2-hydroxy-ethoxy methylguanine, adamantanamine,
5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine
arabinoside, benzoic acid, undecylenic alkanolamide,
ciclopiroxolamine, polyenes, imidazoles, allylamine, thicarbamates,
amphotericin B, butylparaben, clindamycin, econaxole, amrolfine,
butenafine, naftifine, terbinafine, ketoconazole, elubiol,
econazole, econaxole, itraconazole, isoconazole, miconazole,
sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole,
terconazole, butoconazole, thiabendazole, voriconazole,
saperconazole, sertaconazole, fenticonazole, posaconazole,
bifonazole, fluconazole, flutrimazole nystatin, pimaricin,
flucytosine, natamycin, tolnaftate, mafenide dapsone, caspofungin,
actofunicone, griseofulvin, potassium iodide. Gentian Violet,
ciclopirox, ciclopirox olamine, haloprogin, ketoconazole, linalool,
undecylenate, silver sulfadiazine, undecylenic acid, undecylenic
alkanolamide and Carbol-Fuchsin.
[0082] Other drugs may be cytokines and chemokines, particularly
anti-inflammatory cytokines such as IL-4 and IL-10.
[0083] The drugs may be bonded to the oxidized cellulose as
discussed above (i.e., ionically or covalently through the
carboxylic group) or through any other atom on the glucose monomers
(e.g., the hydroxy moieties) via any atom of the drug molecule. In
some embodiments, the drug molecule possesses an atom that may be
used for bonding to the oxidized cellulose (For example a
heteroatom via which binding to the oxidized cellulose may take
place or a pendent group which may directly interact with an atom
or group of the oxidized cellulose). In some other embodiments, the
binding of the drug to the oxidized cellulose necessitates a
chemical group transformation as may be known to a person skilled
in the art.
[0084] In some other embodiments, the drug is associated with the
oxidized cellulose via a non-covalent bonding.
[0085] The compositions of the invention may comprise any
combination of glucans, or oxidized celluloses. In one embodiment,
the composition comprises a single form of the oxidized cellulose,
e.g., only amorphous oxidized cellulose, preferably in the form of
micro or nanoparticles. In another embodiment, the composition
comprises at least two forms of oxidized cellulose, for example
nanoparticles in combination with microparticles. In another
embodiment, the composition comprises oxidized cellulose and an
oxidized cellulose derivative.
[0086] In some embodiments, the at least one glucan, e.g., oxidized
cellulose may be part of a core-shell system, a lipid vesicle, a
liposome, or any other carrier having an inner core containing the
oxidized cellulose and optionally at least one additional additive
or solvent, and a shell which substantially surrounds it.
Alternatively, the core may contain optionally an additive or a
solvent and the shell which substantially surrounds it may be the
glucan, a salt or derivative thereof.
[0087] The pharmaceutical composition of the present invention may
further comprise at least one drug or therapeutics as detailed
hereinbefore and any other additive as disclosed hereinbelow.
[0088] The compositions of the present invention may be adapted
and/or packaged as a kit for personal use or for use by a medical
practitioner, for periodic administration to a subject in doses
over any period of time.
[0089] Typically, for the prevention or treatment of allergies and
related diseases or disorders, the period is of 3-30 days, in doses
at least once daily up to ten times/day. The composition may for
example contain in each one of said doses up to 2 g of the glucan,
e.g., oxidized cellulose, salt or a derivative thereof. In another
example, dosages of about 200-1,000 mg of the glucan, adapted for
administration to said subject may be prepared in capsules,
tablets, lozenges, as a powder, a suspension, syrup, a cream, eye
or nasal drops or ointment, etc.
[0090] As used herein, the term "subject" includes animals,
particularly mammalian animals, and most preferably humans.
Non-human animals include for example primates, domestic animals,
farm animals, and experimental animals. The term also encompasses
all animal disease models for e.g., asthma and allergy and
naturally occurring or non-naturally occurring mutated or non-human
genetically engineered (e.g., transgenic or knockout) animals.
[0091] As stated hereinbefore, the compositions of the invention
are used for the treatment and/or prophylaxis of at least one
disease or disorder associated with at least one allergen. The term
"allergen" refers to all foreign agents capable of inducing,
promoting, or stimulating allergy, i.e., the hypersensitive state
induced by an exaggerated immune response to the allergen, or
asthmatic reaction in a subject. The term encompasses plant/tree
pollens or spores, animal dander, house dust mite, dust, lint, mite
feces, fungal spores, and cockroaches. In one preferred embodiment,
said allergen is pollen.
[0092] Non-limiting examples of plant or tree pollen include:
[0093] weed pollen such as, but not limiting to, dandelion,
goldenrod, nettle, sage, clover, ragweed, mugwort, pellitory,
nettles and dock; [0094] grass pollen such as, but not limiting to,
Bermuda couch grass, sweet vernal grass, red and blue grasses,
Johnson grass pollen; ryegrass such as Italian or annual ryegrass,
perennial ryegrass, hybrid ryegrass, timothy grass, orchard grass,
tall fescue, meadow fescue and red fescue; [0095] tree pollen such
as, but not limiting to, alder, oak, ash, cypress, olive, maple,
cedar, western red cedar, elm, birch, hickory, poplar, American
sycamore, and walnut; and [0096] annual plant pollen such as, but
not limiting to, tobacco and cotton.
[0097] Animal allergens may for example be skin, hair, various
parasites and fungi. In one particular embodiment, the animal
allergen is associated with cat allergens.
[0098] As may be known to the person skilled in the art, when the
allergen enters the respiratory system or when coming in contact
with the skin or eyes it induces an array of diseases or disorders
which may be directly caused by the allergen, and thus is referred
to as a "disease or disorder mediated by" the allergen.
Alternatively, the disease or disorder, although initially not
mediated by the allergen, may deteriorate due to exposure to an
allergen. Limiting or avoiding contact of a tissue with said
allergen may prevent or lessen such disease or disorder. Such a
disease or disorder which deteriorates by allergen contact is
referred to as a disease or disorder which is "associated with at
least one allergen.". In its broadest definition, this expression
is used to mean that there exists a relationship between an
exposure to an allergen and the induction of a symptom, a
condition, a disorder or a disease, or that there exists a
secondary effect of the exposure to the allergen which exacerbates
a condition, a disorder or a disease that may have been initially
caused by another factor. One association may for example be
between a disease which is either caused by binding of IgE to an
allergen causing immediate Type I allergic reaction or to a disease
that is caused by another factor aggravated by the IgE-allergen
interaction. A symptom that is present in a subject may therefore
be the direct result of or caused by the exposure to the
allergen.
[0099] Examples of such a disease or a disorder are the
inflammatory, allergic and non-allergic diseases or disorders of
the respiratory system or the skin. An inflammatory condition,
disorder or disease refers to one or more physiological responses
that characterize or constitute inflammation. An allergy or
allergic condition, as used herein refers to a hypersensitivity to
an allergen. Such conditions, disorders and diseases include but
are not limited to allergic asthma, asthma, extrinsic bronchial
asthma, chronic obstructive pulmonary disease, hay fever (seasonal
rhinitis), allergic rhinitis, allergic conjunctivitis, hives,
eczema, urticaria, angioedema, onchocercal dermatitis, atopic
dermatitis, dermatitis, swelling, hypersensitivity pneumonitis and
bronchopulmonary dysplasia.
[0100] In one embodiment, the disease or disorder associated with
at least one allergen is allergic rhinitis, allergic
conjunctivitis, hives, hay fever and asthma of any type and in
particular seasonal asthma, allergic skin diseases including
urticaria, angioedema and atopic dermatitis. In another embodiment,
the disease or disorder is asthma or allergic rhinitis.
[0101] Asthma refers herein to an allergic or non-allergic
condition, disorder or disease of the respiratory system that is
episodic and characterized by inflammation with constriction,
narrowing or obstruction of the airways. Allergic asthma is
typically associated with increased reactivity of respiratory
system to an inhaled allergen. Asthma is frequently, although not
exclusively associated with atopic or allergic symptoms. Typically,
a subject with asthma suffers from recurrent attacks of cough,
shortness of breath with wheezing, chest pain, chest tightness,
etc. While a plurality of such adverse symptoms typically occur in
asthma, the existence of any one is usually adequate for diagnosis
of asthma, and for treatment in accordance with the invention.
Asthmatic conditions can be acute, chronic, mild, moderate or
severe asthma (unstable asthma), nocturnal asthma or asthma
associated with psychological stress.
[0102] Allergic rhinitis is an allergic reaction of the nasal
mucosa (upper airways), which includes hay fever (seasonal allergic
rhinitis) and perennial rhinitis (non-seasonal allergic rhinitis)
which are typically characterized by seasonal or perennial
sneezing, rhinorrhea, nasal congestion, pruritis and eye itching,
redness and tearing.
[0103] The composition may also comprise a pharmaceutically
acceptable carrier, such as a vehicle, an adjuvant, an excipient,
or a diluent. Such carriers are well known to those who are skilled
in the art and are readily available to the public. It is preferred
that the pharmaceutically acceptable carrier be one which is
chemically inert to the glucan or oxidized cellulose or any other
component of the composition and one which has no detrimental side
effects or toxicity under the conditions of use.
[0104] The choice of carrier will be determined in part by the
particular composition, as well as by the particular method used to
administer the composition. Accordingly, there is a wide variety of
suitable formulations of the pharmaceutical composition of the
present invention. The following formulations for topical, oral,
aerosol, intranasal, intraocular, parenteral, subcutaneous,
intravenous, intramuscular, interperitoneal, rectal, and vaginal
administrations are merely exemplary and are in no way
limiting.
[0105] Formulations for topical application on the skin of the
subject or on the subject's hair may be in the form of a gel,
ointment, emulsion, thick cream, liniment, balsam, lotion, foam,
mask, shampoo, tonic means, cleaner, spray, hair spray, (or it may
be in the form of a means for the hair treatment such as rinsing,
coloring, discoloring, hairdressing, hair straitening, hair waving,
or hair fixing), powder including liquid powder, compact powder,
cosmetic pencil, or in any other traditional form used in the field
of cosmetology or dermatology.
[0106] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the oxidized
cellulose dissolved in diluents, such as water, saline, or orange
juice; (b) capsules, sachets, tablets, lozenges, and troches, each
containing a predetermined amount of the oxidized cellulose, as
solids or granules; (c) powders; (d) suspensions in an appropriate
liquid; and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending
agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers, such as lactose,
sucrose, calcium phosphate, and starch. Tablet forms can include
one or more of lactose, sucrose, mannitol, corn starch, potato
starch, alginic acid, acacia, gelatin, guar gum, colloidal silicon
dioxide, croscarmellose sodiumk talc, magnesium stearate, calcium
stearate, zinc stearate, stearic acid, and other excipients,
colorants, diluents, buffering agents, disintegrating agents,
moistening agents, preservatives, flavoring agents, and
pharmacologically compatible carriers. Lozenge forms can comprise
the oxidized cellulose in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to
the oxidized cellulose, such carriers as are known in the art.
[0107] The compounds of the present invention, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation or
intranasally. These aerosol formulations can be placed into
pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
may be formulated as pharmaceuticals for non-pressured
preparations, such as in a nebulizer or an atomizer.
[0108] Formulations for intranasal or mucosal delivery may comprise
enhancing agents such as solubilization agents; charge modifying
agents; pH control agents; degradative enzyme inhibitors; mucolytic
or mucus clearing agents; ciliostatic agents; membrane
penetration-enhancing agents such as surfactants, bile salts,
phospholipid or fatty acid additives, mixed micelle, liposome, or
carrier, alcohols, enamines, NO donor compounds, long-chain
amphipathic molecules, small hydrophobic penetration enhancers;
sodium or a salicylic acid derivatives, glycerol ester of
acetoacetic acids, cyclodextrin or beta-cyclodextrin derivatives
medium-chain tatty acids, chelating agents, amino acids or salts
thereof, N-acetylamino acids or salts thereof, enzyme degradatives
to a selected membrane component, inhibitors of fatty acid
synthesis, inhibitors of cholesterol synthesis; or any combination
of these membrane penetration enhancing agents; modulatory agents
of epithelial junction physiology, such as nitric oxide (NO)
stimulators, chitosan, and chitosan derivatives; vasodilator
agents; selective transport-enhancing agents; and stabilizing
delivery vehicles, carriers, supports or complex-forming species
which is/are effectively combined, associated, contained,
encapsulated or bound to stabilize the oxidized cellulose for
enhanced mucosal delivery.
[0109] In some embodiments of the invention, the mucosal
therapeutic and prophylactic compositions of the present invention
may be supplemented with any suitable penetration-promoting agent
that facilitates absorption, diffusion, or penetration of the
composition or any component thereof across mucosal barriers.
[0110] Certain formulations for intranasal applications as for
aerosol applications are specifically adapted for a selected target
cell, tissue or organ, which are at a remote target site or even a
particular disease state. Efficiently loaded formulations at
effective concentration levels in a carrier or other delivery
vehicle, may be delivered and maintained in a stabilized form,
e.g., at the nasal mucosa and/or during passage through
intracellular compartments and membranes, to a remote target site
for action (e.g., a defined tissue, organ, or extracellular
compartment).
[0111] Formulations for intraocular administration may be
administered topically to the eye or eye lid, for example, using
drops, an ointment, a cream, a gel, a suspension, etc. The oxidized
cellulose may be formulated with excipients such as
methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl
cellulose, polyvinyl pyrrolidine, neutral poly (meth)acrylate
esters, and other viscosity-enhancing agents.
[0112] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions
vehicle can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The oxidized cellulose can be
administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol, isopropanol, or
hexadecyl alcohol, glycols, such as propylene glycol or
polyethylene glycol, glycerol ketals, such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester
or glyceride, or an acetylated fatty acid glyceride with or without
the addition of a pharmaceutically acceptable surfactant, such as a
soap or a detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0113] Oils, which can be used in parenteral formulations, include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters. Suitable soaps for use in parenteral formulations
include fatty alkali metal, ammonium, and triethanolamine salts,
and suitable detergents include (a) cationic detergents such as,
for example, dimethyl dialkyl ammonium halides, and alkyl
pyridinium halides, (b) anionic detergents such as, for example,
alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and sulfosuccinates, (c) nonionic
detergents such as, for example, fatty amine oxides, fatty acid
alkanolamides, and polyoxy-ethylenepolypropylene copolymers, (d)
amphoteric detergents such as, for example,
alkyl-.beta.-aminopriopionates, and 2-alkyl-imidazoline quaternary
ammonium salts and (3) mixtures thereof.
[0114] The parenteral formulations will typically contain from
about 0.05 to about 25% by weight of the oxidized cellulose in
solution. Suitable preservatives and buffers can be used in such
formulations. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophile-lipophile balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5 to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such
as sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose scaled
containers, such as ampoules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0115] The compositions of the present invention may be made into
injectable no formulations. The requirements for effective
pharmaceutical carriers for injectable compositions are well known
to those of ordinary skill in the art. See for example
Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co.,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982),
and ASHP Handbook on the Injectable Drugs, Toissel, 4th ed., pages
622-630 (1986).
[0116] Additionally, the compositions of the present invention may
be made into suppositories by mixing with a variety of bases, such
as emulsifying bases or water-soluble bases. Formulations suitable
for vaginal administration may be presented as pessaries, tampons,
creams, gels, pastes, foams, or spray formulas containing, in
addition to the active ingredient, such carriers as are known in
the art to be appropriate.
[0117] In one embodiment of the invention, the composition is a
topical composition formulated for administration onto the skin
(including eyes, scull), hair and nails) of a subject.
[0118] In another embodiment, the composition is formulated for
mucosal application.
[0119] In another embodiment, the composition is Formulated for
oral administration.
[0120] In another embodiment, the composition is formulated for
intraocular administration e.g., as eye drops, creme, etc.
[0121] In another embodiment, the composition is formulated for
intranasal administration.
[0122] In another embodiment, the composition is formulated for
inhalation.
[0123] Subjects predisposed or susceptible to, exposed to or
allergic to allergens such as those disclosed herein may be at risk
of having asthma and, therefore, are amenable to treatment in
accordance with the invention. The compositions of the invention
are also useful for contacting with or administering to subjects
prophylactically, namely prior to manifestation or onset of asthma
or any other condition or an associated symptom or physiological or
psychological response, such that it can eliminate, prevent,
inhibit, decrease or reduce the probability, susceptibility or
frequency of having asthma or an associated symptom.
[0124] In another aspect of the present invention, there is
provided a method for the treatment or prophylaxis of at least one
disease or disorder mediated by or associated with at least one
allergen, said method comprising administering to a subject an
effective amount of at least one glucan. In one embodiment, said at
least one glucan is oxidized cellulose, a salt or derivative
thereof.
[0125] As stated hereinbefore, the glucan is not cellulose or any
non-oxidized form thereof.
[0126] In one embodiment of the present invention, said subject is
one suffering from said disease or disorder.
[0127] In another embodiment, said subject has a predisposition to
said disease or disorder. The predisposition may be genetic or
environmental.
[0128] In another embodiment, said at least one disease or disorder
is an allergic reaction.
[0129] In another aspect of the present invention, there is
provided method for delaying the onset or lessening the severity of
at least one allergic reaction, said method comprising
administering to a subject an effective amount of at least one
glucan. In one embodiment, said at least one glucan is oxidized
cellulose, a salt or derivative thereof.
[0130] The invention further provides a method for reducing a
subject's sensitivity to at least one allergen, said method
comprising administering to said subject an effective amount of at
least one glucan. In one embodiment, said at least one glucan is
oxidized cellulose, a salt or derivative thereof.
[0131] The invention further provides a method of reducing a
subject's sensitivity to a pollen allergen in a subject sensitive
to such pollen allergen or a second allergen immunologically
cross-reactive with said pollen allergen, comprising administering
to said subject at least one glucan. In one embodiment, said at
least one glucan is oxidized cellulose, or a salt or derivative
thereof.
[0132] The methods and compositions of the present invention are
appropriate for treatment of subjects exposed to an allergen or who
are susceptible to having an allergic reaction. Within the scope of
the present invention, subjects who are at risk of having an
allergic reaction include subjects having a predisposition towards
an allergic reaction, or infection or exposure to an agent that is
associated with an allergy or allergic reaction due to a genetic or
environmental risk factor. Subjects having a predisposition can be
identified by a personal or family history, through genetic
screening, tests appropriate for detection of increased risk, or
exhibiting relevant symptoms indicating predisposition or
susceptibility. The allergic reaction may be any such symptom or
condition associated with, for example, an existing allergic
condition, a symptom or condition associated with or caused by an
allergic condition, an acute allergic episode, a latent allergic
condition, and seasonal or geographical tendencies.
[0133] The methods of the invention are directed at providing a
beneficial effect or therapeutic benefit to a subject, either
short-term and/or longer-term. Thus, the term "treatment" or any
lingual variation thereof, refers within the scope of the present
invention to a clinical endpoint characterized by an improvement in
the subjects condition; a reduction in the severity, frequency,
duration or progression of one or more adverse symptoms or
complications associated with the disease or disorder; and/or an
inhibition, reduction, elimination, prevention or reversal of one
or more of the physiological, biochemical or cellular
manifestations or characteristics of the disorder or disease,
including complete prevention of the disease or disorder.
[0134] Thus, in one embodiment of the methods of the invention, the
treatment and/or prophylaxis of at least one disease or disorder
associated with at least one allergen comprises reducing
progression, severity, frequency, duration, susceptibility or
probability of inflammatory, allergic and non-allergic conditions,
disorders and diseases of a subject suffering from any one of such
conditions or having a genetic or environmental predisposition to
having one or more of the conditions, symptoms, diseases or
disorders.
[0135] In one embodiment, the disease or disorder is asthma and the
condition or symptom is any one symptom associated with asthma.
[0136] In order to achieve any one of the therapeutic benefits of
the compositions and methods of the invention, the active
component, namely the at least one glucan, being preferably an
oxidized cellulose, a salt or a derivative thereof, should be
administered therapeutically or prophylactically in an efficient
amount which may vary according to the status of the condition, the
type of treatment sought (i.e., therapeutic or preventive), the
general condition of the subject, the use of other drug or agent
and any other factor as may be known to a medical practitioner. The
dose amount, frequency or duration of administration may be
proportionally increased or reduced. The term "effective amount" or
any lingual variation thereof, refers generally to a therapeutic or
prophylactic amount which is, when administered to a subject,
sufficient to reduce, prevents delay and/or inhibit the onset or
progression or worsening of a disease or disorder; to reduce
relieve, and/or alleviate the severity, frequency, duration,
susceptibility or probability of one or more undesirable symptom or
condition associated with the disease or disorder; to hasten the
recovery from one or more symptoms associated with the disease or
disorder.
[0137] The treatment or prophylactic regimens may be short term or
long term and may depend on such factors as discussed hereinabove.
The compositions or methods of the invention may employ a single
administration of any one composition or multiple administrations,
wherein the composition is administered alone or in combination
with other therapeutics or treatments.
[0138] Without wishing to be bound to specific dosages and
particular regimes, as the therapeutic or prophylactic efficacy of
the compositions and methods of the invention may vary between one
subject to another, a subject may be administered a composition of
the invention once, twice, three, four, five or more times daily,
weekly, monthly or annually. Depending on the therapeutic effect
sought, therapeutic or a prophylactic, and the type of formulation,
e.g., for oral, nasal or topical administration, the dose size may
vary between about 0.1 mg/kg, to about 100 mg/kg.
[0139] The composition of the invention may be administered by a
medical practitioner or by the subject being treated prior to an
expected contact with an allergen, immediately after such a
contact, or within a short period after the onset of at least one
symptom associated with a disease or disorder.
[0140] Apart from the manufacture of the pharmaceutical
compositions of the invention, according to another aspect of the
present invention, the at least one glucan, and most preferably the
oxidized cellulose, a salt or derivative thereof is also used for
the preparation of compositions or formulations for non-therapeutic
purposes having to do with the reduction or complete elimination of
a plurality of allergen from an environment.
[0141] According to this aspect of the invention the at least one
glucan, preferably the oxidized cellulose, a salt or derivative
thereof, may be employed as an allergen inactivator in the
manufacture of such paper products such as a mask, a sheet; air
filtering units; cosmetic products such as a creme, a spray, an
aerosol; household solutions such as a detergent, a laundering
agent; etc. The at least one glucan may be embedded in the material
from which the object is prepared or may be applied thereon by
distributing, spraying, coating, or evaporating a solution or
formulation containing thereof. The allergen inactivating
formulation may be applied to any surface suspected of having to
thereon allergens.
BRIEF DESCRIPTION OF THE DRAWINGS
[0142] in order to understand the invention and to see how it may
be carried out in practice, embodiments will now be described, by
way of non-limiting example only, with reference to the
accompanying drawings, in which:
[0143] FIGS. 1A-1D demonstrates the efficacy of the compositions of
the present invention in animal model. BALB/c mice (3 groups) were
sensitized intranasally with 100 .mu.g/animal of grass pollen
soluble proteins in 50 .mu.l PBS on days 0, 1, and 2 and challenged
on days 14, 15, 18, and 19 intranasally with 25 .mu.g grass pollen
soluble proteins. A naive control was made by intranasally treating
BALB/c mice with PBS. On day 21 mice one group of asthmatic mice
and the naive mice were sacrificed and BALF were obtained. Total
BALF cells were counted (FIG. 1A) and cells were stained by
May-Grunwald Giemsa and were characterized morphologically (FIG.
1B). On day 26 the remaining two asthmatic groups received a
challenge by inhalation of either pollen proteins only or pollen
proteins with OCAM. Mice were sacrificed at day 29 and BALF were
obtained. Total BALF cells were counted (FIG. 1C) and cells were
stained by May-Grunwald Gieemsa and were characterized
morphologically (FIG. 1D).
[0144] FIGS. 2A and 2B demonstrate the specific binding of
microcrystalline non-oxidized cellulose to major rye grass
allergens. Soluble pollen proteins were bound to either KOH-washed
or untreated non-oxidized cellulose particles. Both bound (labeled
B) and unbound (labeled UB) fractions (20 .mu.L), were separated on
a 4-20% glycine gradient gel or 150% glycine gel. Gels were stained
with either coomasie stain (FIG. 2A) or Silver stain (FIG. 2B).
[0145] FIGS. 3A and 3B demonstrate that oxidation of
microcrystalline cellulose into oxidized cellulose increases
dramatically its ability to bind rye grass pollen allergens.
Soluble pollen proteins from either Lolium perenne (FIG. 3A) or
Lolium rigidum (FIG. 3B) were bound to either oxidized cellulose
(prepared from Avicel microcrystalline cellulose) or to
non-oxidized Avicel microcrystalline cellulose. Bound (labeled B)
and unbound (labeled UB) fractions and total proteins (labeled T)
were separated by SDS-PAGE and stained by coomassie.
[0146] FIG. 4 demonstrates the binding of beta-glucan to rye-grass
pollen proteins. Soluble pollen proteins were bound to different
quantities of beta-glucan. Both bound (labeled B) and unbound
(labeled UB) fractions were separated by 15% glycine gel and
stained with silver stain.
[0147] FIGS. 5A-5C illustrate the general concept of a
oxidized-cellulose blocking mechanism.
[0148] FIGS. 6A and 6B illustrate an exemplary application of a
composition of the invention by inhalation.
DETAILED DESCRIPTION OF EMBODIMENTS
[0149] As disclosed herein, glucans and particularly oxidized
cellulose have the necessary recognition of various plants
allergens and the capability of binding thereto. Without wishing to
be bound by theory and as illustrated for the sake of general
understanding of the invention in FIGS. 5A, 5B and 5C, pollen
allergens from groups I, II, or III bound by specific IgE
antibodies of allergic patients can induce an allergic reaction
(FIG. 5A). Pollen allergens can also bind glucans and
polysaccharides such as oxidized cellulose through there CBD,
cellulose binding domain (FIG. 5B). Since most of the IgE binding
domains of pollen allergens of groups I, II and III are found in
their CBD, polysaccharides such as oxidized cellulose can compete
with IgE specific antibodies on allergen binding and thereby
inhibit the allergic reaction. The employment of pharmaceutical
compositions comprising such glucans by patients by various
application methods such as by spraying the composition onto the
skin or tissue (FIG. 5C), assists in the formation of a reactive
barrier that prevents the association of the allergen and the
antibodies.
[0150] As FIGS. 6A and 6B further illustrate, allergen specific IgE
antibodies may encounter pollen allergens of group I, II, or III in
the bronchi of allergic patients and thus induce an allergic
reaction (FIG. 6A). The application of glucans such as to oxidized
cellulose to the patient's bronchi prevents the binding of pollen
allergen to IgE specific antibodies and thereby inhibits the
allergic reaction (FIG. 6B). It has been demonstrated that the
glucans can prevent allergen binding to IgE either through direct
competition or by creation of a relatively large allergen-glucan
complex that is removed from the bronchi, through mucociliary
transport.
[0151] While FIGS. 5A-5C and 6A-6B illustrate specifically oxidized
cellulose and application thereof by inhalation, it should be
understood that this example is provided as a single illustration
and should not be taken to be any limitation of scope. The
compositions of the invention may be applied to a target organ or
tissue by ally method known and in any dosage as may be
necessitated by the condition, the severity thereof, the subject,
and other parameters as known to a medical practitioner.
EXAMPLES
Example 1
Preparation of Various Oxidized Cellulose Amorphous Microparticles
(OCAMs)
[0152] Different Oxidized Cellulose Amorphous Microparticles
(OCAMs) with different degree of crystallinity and oxidation were
prepared essentially as described by Kumar et al (2002).
[0153] Briefly, nitric acid and phosphoric acid were mixed in 4:1
(v/v) ratios. To a 140 ml solution of the acid mixture, 10.0 g of
microcrystalline cellulose was added. Once the cellulose was
completely soaked. 2.0 g of sodium nitrite were added in one
portion. An immediate formation of reddish brown fumes occurred.
The reaction mixture was allowed to react at room temperature. With
occasional stirring, for 24 hours. The reaction mixture, which
appeared green in color, was terminated by adding 700 ml of doubly
distilled water (DDW). The diluted reaction mixture was filtered,
and the white fluffy solid obtained was washed with water until the
filtrate showed a pH of about 4. The solid was washed with acetone
and then air-dried at 60.degree. C. and at room temperature. The
dried oxidized cellulose was milled to 10 microns by a vortexmill
(Super Fine, Israel). The distribution of the particles size was
determined by MASTERSIZER 2000 MALVERN (England) and was from 1-10
microns.
[0154] The particle size can be changed by milling to different
sizes using, any one milling apparatus. Size and shape were
determined by light and electron microscopy. Degree of
crystallinity was determined by X-ray diffraction.
[0155] In some embodiments of the invention, the thus prepared
OCAMs may be reacted with various glucans by mixing the OCAMs with
a solution of the glucan. The glucan may be one or more selected
amongst rye flour arabinoxylan barely beta-glucan, oat beta-glucan
galacton (Lupin), pullulan, carob galactomannan, xyloglucan, guar
galactomannan, pectic galactan, rhamnogalacturonan-galacturonic
acid, pachyman, curdlan, and mannan. The glucans adsorb to the
cellulose by non-covalent bonding. After binding, the unbound
material is removed and the particles are dried to remove the
remaining solvent.
[0156] In other embodiments, the particles are of chitosan and not
oxidized cellulose.
Example 2
Determination of Optimal Allergen Blocking OCAMs
[0157] Different preparations of OCAMs are used in-vitro to block
different allergen preparations and prevent IgE binding of sera
from allergic patients. Aqueous solutions containing different
pollen protein allergens are incubated with different OCAMs. The
particles are removed by centrifugation and the resulted proteins
are tested for IgE binding using ELISA. OCAMs that show the highest
IgE blocking activity are used for further analysis. Histamine
release is performed with heparinized whole blood as described by
Shirai et al., (1997). The patients, men and women with
pollen-induced asthma, who had been diagnosed by skin test and
inhalation challenge are treated. As controls are chosen asthmatic
subjects with no response to pollen allergen.
[0158] Heparinized whole blood samples are taken and incubated with
pollen allergens in the presence or absence of selected OCAMs at
various concentrations for 30 minutes at 37.degree. C. After
centrifugation, histamine levels are measured in the cell-free
supernatants by histamine ELISA kit (IBL, Hamburg Germany).
Histamine release is expressed as a percentage of total histamine.
Successful OCAMs are chosen based on their ability to block wide
spectrum of allergens and reduce or even prevent histamine
release.
Example 3
Determination of Allergy Blocking by OCAM in an Animal Model
[0159] The following is a basic protocol developed for determining
the efficacy of the compositions and methods of the invention on
animals. A person skilled in the art would appreciate that this
protocol may be regarded of only one example and that other more
specific or more generic models may be developed for the
purpose.
[0160] In the following, female, 8-9 week old BALB/c mice are used
for asthma model. Mice are divided into 3 treatment groups (naive,
asthma, and treatment groups) 10 mice in each group. Naive mice are
used as negative controls and are challenged with normal saline
instead of the grass pollen allergen. 20 mice are sensitized as
described below. 10 mice are treated with OCAM inhalation before
each allergen challenge (treatment group).
[0161] Grass pollen is used for the preparation of an extract. Five
grams of pollen are suspended in 50 ml phosphate buffered saline
(PBS) and extracted by stirring at room temperature for 15 minutes.
After centrifugation at 20000.times.g for 10 min at 4.degree. C.
the supernatant is filtered through a 0.45-micron filter. The
protein concentration of the grass pollen extract is determined
using the BCA reagent (Pierce).
[0162] For sensitization 100 .mu.g/animal grass pollen adsorbed to
Al(OH).sub.3 (2 mg/animal) is injected intraperitoneally (i.p.)
three times in 14-day intervals. Animals are challenged by either
allergen inhalation (1 mg/ml) for 10 minutes each time with the
mice placed unrestrained in a 20-liter box connected to an
ultrasonic nebulizer (LS 230 System Villeneuve Sur Lot, France) or
intranasal administration (25 .mu.g/animal) in 50 .mu.l PBS on day
0, 3 and 6 after the last injection. Alternatively, mice can be
challenged once or twice at days 20-24.
[0163] Bronchoconstriction is measured at any of the days of the
challenge or in the days that follow the 6 day challenge by a
modified noninvasive method (Hamelmann et al. 1997) using
barometric plethysmography and expressed as the enhanced pause
(Penh), a calculated dimensionless value that correlates with
measurement of airway resistance, impedance, and intra-pleural
pressure. Penh is measured before (as baseline) and after each
allergen challenge. Data is expressed as the percent change of Penh
compared to baseline (% Penh).
[0164] Mice are sacrificed under deep anesthesia with 2,2,2
tribromoethanol solution 24-72 hours after the last challenge. A
midline celiotomy is performed, and the animals are euthanized by
exsanguination by withdrawal of blood from the inferior vena cava.
Serum is separated from the blood and used to determine total and
allergen specific levels of 1-E and IgG in the blood. A 21-gauge
needle is inserted into the trachea and secured with a 3-0 silk
suture. The lungs are lavaged via the tracheal needle with
3.times.1 ml of PBS. Lavage fluid is later used for morphological
characterization of cells on cytospin slides after May-Grunwald
Giemsa staining for characterization of cells by FACS analysis and
for examination of cytokines levels by ELISA. The lungs are removed
and inflated with 4% buffered formalin under pressure of 20 cm
H.sub.2O The tissues are embedded in paraffin and 2 to 3 .mu.m
sections are cut and stained with H&E for viewing by light
microscopy the inflammatory changes in the peribronchial areas. The
serum is collected for IgE level measurements.
[0165] Data is collected on an excel spreadsheet. The differences
between groups are calculated with ANOVA test for the parametric
measurements and with Kruskal Wallis test for the ordinal
parameters (pathology score). A p<0.05 is considered
significant.
Example 4
Animal Model
[0166] In the following, female, 8-9 week old BALB/c mice were used
for the development of an asthma model. The experiment was divided
into two parts:
[0167] In the first part establishment of rye grass pollen allergic
mice model was achieved through intranasal sensitization and
challenge with Lolium perenne pollen extract in a 21-day time
period. In this part there were two groups of mice: asthmatic,
which received both sensitization and challenge with Lolium perenne
pollen extract and naive which received both sensitization and
challenge with PBS. Each group contained 3 mice.
[0168] In the second part all the mice were sensitized and
challenged with Lolium perenne pollen extract for 21 days. As day
26, the mice were divided into two groups: asthma group which
received a 15 minute inhalation of Lolium perenne pollen extract
and the treatment group which received a 15 minute inhalation of
Lolium perenne pollen extract mixed with OCAM. Each group contained
3 mice.
[0169] Lolium perenne (Perenial rye grass), Batch 021405105
obtained from Allergon A B, Sweden, was used for the preparation of
an extract. Five grams of pollen were suspended in 50 ml phosphate
buffered saline (PBS) and extracted by stirring at room temperature
for 15 minutes. After centrifugation at 20000.times.g for 10 min at
4.degree. C. the supernatant was kept and the content of soluble
proteins was determined using the BCA reagent (Pierce).
[0170] Sensitization--Mice were anesthetized with isoflurane and
then sensitized intranasally with 100 .mu.g/animal of grass pollen
soluble proteins in 50 .mu.l PBS on days 0, 1, and 2.
[0171] Airway Challenge--Mice were anesthetized with isoflurane and
challenged on days 14, 15, 18, and 19 intranasally with 25 .mu.g
grass pollen soluble proteins. Naive mice were sensitized and
challenged intranasally with PBS. The mice were then either
sacrificed at clay 21 or given an additional 15-minute inhalation
challenge of either grass pollen soluble proteins 4 mg/ml or grass
pollen soluble proteins 4 mg/ml OCAM mixture (4 mg/ml pollen
proteins and 13 mg/ml OCAM) at day 26 and sacrificed at day 29.
[0172] Analysis of Bronchoalveolar Lavage (BAL)--Bronchoalveolar
lavage fluids (BALF) were obtained via cannulation of the exposed
trachea, by infusion of 3.times.1 ml of PBS through a 21-gauge
needle into the lungs, followed by aspiration of this fluid into a
syringe. Aliquots were centrifuged, and supernatants were collected
and stored at -80.degree. C. for future cytokine analysis. Cell
pellets were counted and subjected to cytospin, and the slides were
stained May-Grunwald Giemsa and were characterized
morphologically.
[0173] Sensitization and challenge with Rye grass pollen extract
results in allergic lung inflammation of BALB/C mice--Since there
is no available model of Rye grass pollen allergic mice a mouse
model was developed using one of the accepted methods. BALB/c mice
were thus sensitized intranasally with 100 .mu.g/animal of grass
pollen proteins extract in 50 .mu.l PBS on days 0, 1, and 2 and
challenged on days 14, 15, 18, and 19 intranasally with 25 .mu.g
grass pollen soluble proteins. On day 21 mice were sacrificed and
BALF were obtained and analyzed.
[0174] As FIG. 1A shows, the asthmatic animals that were treated
with pollen proteins, showed an increase in their BALF cell counts
compared to the naive group that was treated with PBS, indicating
the development of lung inflammation. Since the allergic reaction
is characterized by Th2 mediated lung inflammation the nature of
cells in the BALF was next evaluated by May-Grunwald staining. As
FIG. 1B shows there was a significant increase in the recruitment
of eosinophils to the lungs in the asthmatic mice compared with the
naive mice indicating that the inflammation was indeed of allergic
nature.
[0175] OCAM can attenuate the induction of allergic inflammation in
asthmatic mice--It was next needed to verily whether OCAM could
block the initiation of a secondary lung inflammation in the
asthmatic mice. The asthmatic mice reached a peak of inflammation
of the lungs at day 21 and were thus left untreated until day 26 in
order to allow the inflammation to calm down. At day 26 the
asthmatic mice were divided into two groups and both were given a
challenge by inhalation. However, one group received a challenge of
grass pollen protein extract only while the second group received
an inhalation of OCAM grass pollen extract mixture. The mice were
sacrificed 72 hours later and their cell counts and morphology were
analyzed in the BALF. As can be seen in FIG. 1C the total number of
cells was quite low when compared to the inflammation at day 21,
with no significant difference between the two groups, indicating
that the single challenge at day 26 induced only a weak
inflammation. Examination of the nature of the cells in the BALF,
however, showed a reduction in the percent of macrophages (the main
cells in healthy mice) and an increase in the percent of
eosinophils and lymphocytes in the pollen only group, indicating
the induction of inflammation by the allergic challenge.
Importantly, the change in all the parameters of inflammation that
were just described was significantly lower in the group that
received the OCAM and grass pollen protein mixture (FIG. 1D). This
clearly and unambiguously indicated that the binding of pollen
allergens to OCAM prevented them from inducing the allergic
reaction.
[0176] In similar experiments, particles of chitosan are used in
place of the oxidized cellulose.
Example 5
Determination of Allergy Blocking by OCAM in Rat Animal Model
[0177] The following is a basic protocol developed for determining
the efficacy of the compositions and methods of the invention on
animals. A person skilled in the art would appreciate that this
protocol may be regarded of only one example and that other more
specific or more generic models may be developed for the
purpose.
[0178] In the following, male, Brown Norway rats, 3 weeks of age
and weighing 150 grams each are used for an asthma model. Rats are
divided into 3 treatment groups (naive, asthma, and treatment
groups), 10 rats in each group. Naive rats are used as negative
controls and are not sensitized. 20 rats are sensitized as
described below. 10 rats are treated with OCAM inhalation before
each allergen challenge (treatment group).
[0179] Grass pollen is used for the preparation of an extract. Five
grams of pollen are suspended in 50 ml phosphate buffered saline
(PBS) and extracted by stirring at room temperature for 15 minutes.
After centrifugation at 20000.times.g for 10 min at 4.degree. C.
the supernatant is filtered through a 0.45-micron filter. The
protein concentration of the grass pollen extract is determined
using the BCA reagent (Pierce).
[0180] Induction of asthma--Brown Norway rats, 3 weeks old,
weighing 150 grams each are sensitized at day 0 by subcutaneous
injection of 1 mg of Lolium perenne (Rye grass) soluble proteins
and 200 mg of aluminum hydroxide (Merck) in 0.9% (w/w) saline in a
total volume of 1 mL, and intraperitoneal injection of 1 mL saline
containing Bordetella pertussis (6.times.10.sup.9 heat killed
organisms) (Pasteur Marieux, France). The animals are challenged
every other day from day 14 until day 21 with repeated allergen
(Lolium perenne proteins) inhalation 1 mg/ml.
[0181] Bronchoconstriction Measurements--Before and after every
challenge bronchoconstriction is measured in unrestrained conscious
rats using a method validated by (Hamelmann et al., 1997) and
expressed as the enhanced pause (Penh), before and after allergen
or allergen+OCAM challenge. Animals are placed in a whole-body
plethysmograph. Analogue signals from the amplifier are converted
to digital signals by AD card (LPM-16 National Instruments, Austin,
Tex.). Software (System XA, model SFT 1810, Buxco Electronics) is
used to analyze 10 breath signals and calculate the respiratory
rate, and Penh. This %.DELTA.Penh is used to compare the difference
in bronchoconstriction between the treatment groups.
[0182] Bronchoalveolar Lavage (BAL)--BAL is performed 48 hours
after the last challenge. Mice are anesthetized with I.P. injection
of Ketamine/Xylazine (200 mg/kg) and sacrificed by bleeding from
the abdominal aorta. The mice are than tracheotomized and
incanulated through the trachea. BAL is performed with 5 ml of PBS
in aliquots of 1 ml each time. The lavage fluid is collected in
sterile tubes (Falcon) and placed immediately in ice. BAL is used
for cell count and differential cell count (after May-Grunwald
Giemsa staining) and for measurement of cytokines.
[0183] Pathology--Lungs are removed and fixed by inflation with
paraformaldehyde at a pressure of 20 cm H.sub.2O. The lung tissues
are cut longitudinally in three, embedded in paraffin, randomly
sliced and stained with eosin-hematoxylin for assessments of
interstitial and peri-bronchial inflammation. Other slides are
stained with Alcian blue and PAS for epithelial cell mucus
metaplasia.
Example 6
Formulation of OCAM Suitable for Nasal Spray, Inhalation and
Topical Cream
[0184] OCAM formulations for nasal spray, inhalation and topical
cream are prepared based on common and acceptable ingredients. The
stability of the formulations is tested in an accelerated stability
study. OCAM allergen blocking will be determined by IgE-ELISA.
Example 7
Binding of Rye Grass Pollen Proteins to Microcrystalline Cellulose
or to OCAM Prepared Therefrom
[0185] Preparation of soluble pollen proteins--Pollen from Lolium
perenne or Lolium rigidum (100 mg per sample) were dissolved in 1
ml of PBS. The pollen solvent mixture was rotated for 15 minutes
after which point the pollen was separated from the supernatant by
centrifugation.
[0186] Binding assay--Microcrystalline cellulose, KOH washed
microcrystalline cellulose, OCAM or Barely beta-glucan CAS:P-BGBL
(Megazyme, Ireland) were suspended in PBS alt a concentration of 20
mg/ml. A total of 5-20 mg polysaccharide/sample were precipitated
in an eppendorf tube, resuspended in 450 .mu.l of PBS, mixed with
200 .mu.l of pollen supernatant and rotated for 1 hour. The
particles were precipitated and the supernatant termed unbound
fraction (UB) was collected and mixed 1/1 in sample application
buffer (SAB). The cellulose particles pellets were then washed
twice in PBS and resuspended in 30 .mu.l SAB X2 (termed Bound (B)
fraction). Total proteins (termed T) were made by diluting the
pollen sup 1/4 with PBS and then mixing the diluted proteins 1/1
with SAB X2. All samples were boiled. Samples B, UB and T were
loaded to and separated by 15% or gradient glycine gels and stained
with either coomassie blue or silver stain. Indicated bands were
excised from the gel and analyzed by LC-MS/MS.
[0187] As FIGS. 2-4 show:
[0188] 1. microcrystalline cellulose, OCAM (oxidized cellulose made
from microcrystalline cellulose) and beta-glucan were able to bind
a portion of the pollen proteins;
[0189] 2. sequencing of the major microcrystalline bound proteins
revealed mostly allergens including the expansin family; and
[0190] 3. oxidation of microcrystalline cellulose dramatically
increased its pollen-protein binding capacity.
[0191] Unlike microcrystalline cellulose, OCAM bound most of the
pollen proteins, which can be observed from the massive reduction
of proteins in the unbound fraction, Most of the different pollen
proteins were found in the OCAM bound fraction.
* * * * *